1. Field of the Invention
This invention relates to a method and apparatus for the measurement of antigens and antibodies. More particularly, this invention relates to a method of the quantitative measurement of antigens and antibodies by reacting an antigen or antibody or a mixture thereof with the corresponding antibody and/or antigen supported on insoluble carrier particles having minute diameters and irradiating the resulting reaction mixture with light of wavelengths in the near infrared for a photometric determination of the antigen or antibody, and an apparatus for use therein.
2. Description of the Prior Art
There is a continuing need for rapid, accurate, qualitative and quantitative determinations of biologically active substances, e.g., antigens, antibodies, at extremely low concentrations. Today, there is a wide need for determining the presence of drugs in body fluids. In addition, in medical diagnosis, it is frequently important to know the presence of various substances which are synthesized naturally by the body or ingested.
Heretofore it has been known to detect antibodies or antigens semiquantitatively by reacting latex particles on which an antibody or an antigen is supported with a corresponding antigen or antibody on a glass plate and observing visually the agglutination state.
In recent years, the qualitative and quantitative analysis of trace amounts of substances, particularly antigens and antibodies, has become increasingly important in various fields, not only in the medical world, but in the fields of biochemistry, hygienics, epidemiology and the like. This invention is directed to the development of a method for use in such determination.
It has been known to detect antigens and antibodies semiquantitatively by reacting an antigen or antibody with a latex particles sensitized with the corresponding antibody or antigen on a glass plate and observing visually the agglutination state of the latex.
In recent years, it was proposed in the following articles to quantitatively determine antigens and antibodies using the above-mentioned latex particles by supporting the corresponding antibody or antigen on the latex particles to sensitize the latex, reacting the supported antibody or antigen with the antigen or antibody to be determined to agglutinate the latex particles, and measuring the rate of decrease in turbidity of the supernatant of the latex by means of visible rays for the determination of the antigen or antibody utilizing the agglutination phenomena of the latex reagent:
(A) CROATICA CHEMICA ACTA, 42, (1970), p.p. 457-466; and PA0 (B) European Journal of Biochemistry, Vol. 20, No. 4, (1971), p.p. 553-560. PA0 (C) Immunochemistry, Vol. 12, p.p. 349-351 (1975)
Since the method of the above proposal utilizes the measurement of rate of decrease in turbidity in order to determine the antigen or antibody, it is necessary to use an antibody- or antigen-sensitized latex of an extremely low concentration, for example, in the range of 0.007 to 0.028%, to carry out the reaction of the latex and the antigen or antibody in a standing state, to remove any impurity capable of affecting the turbidity from the sample to be tested, and the like. As a result, the above-mentioned method is disadvantageous in that the rate of the antigen-antibody reaction is inevitably decreased, both the precision and the reproducibility are insufficient for a determination method of antigens or antibodies, and that the removal of impurities sometimes requires extremely complicated operations. Accordingly it is difficult to apply the above method to the determination of such antigens as fibrinogen (Fg), human chorionic gonadotropin (hCG) and the like, since they require complicated procedures for the preparation of their reagent and they do not readily cause reproducible agglutination reactions of the latex if they are present in blood or urine which also contains various other substances capable of adversely affecting the reaction.
Also in the article,
it was proposed to determine quantitatively antibodies and antigens by irradiating the above-mentioned agglutinated latex particles with a laser beam and measuring the change in width of spectral lines of the scattered light of the laser beam in order to determine the mean diffusion constant (D) which is closely related to the Brownian motion of the agglutinated particles which in turn is inversely proportional to the size of the agglutinated particles. Also in this method, since the antibody- or antigen-sensitized latex is used in a extremely low concentration, for example, as low as 0.001%, the rate of the antigen-antibody reaction is so decreased that both the precision and the reproducibility become poor. In addition, this method is also disadvantageous in that it requires complicated calculation using the technique of spectrum analysis which in turn requires complicated operations, and that any impurity in the sample must be removed prior to the measurement. Accordingly, this method has not been put into practice as well.
The above paper C also describes that the determination of antigens and antibodies by the turbidity method as reported in the foregoing paper A gives extremely imprecise results (FIG. 2 on page 350 of the same). We formerly accomplished an invention which provides a method and apparatus for the rapid determination of antigens and/or antibodies in a sample to be tested with a high precision and a good reproducibility, and proposed it in our copending Japanese Patent Application No. 97158/76 (which is hereinafter referred to as "our prior filed application"). In one aspect, the invention of our prior filed application resides in a method for determining antigens and antibodies by reacting in a liquid medium an antigen or antibody or a mixture thereof with the corresponding antibody and/or antigen which has been supported on insoluble carrier particles having an average diameter of not greater than 1.6 microns and irradiating the resulting reaction mixture with light having a wavelength in the range of 0.6 to 2.4 microns and longer than the average diameter of said carrier particles by a factor of at least 1.5 in order to determine the absorbance of the reaction mixture.